Inherited and acquired defects in the formation or adult maintenance of cartilage cause many types of skeletal dysplasias and joint degeneration diseases, namely osteoarthritis. Sox9 encodes a transcription factor that is absolutely required for cartilage formation. The mechanisms underlying Sox9 transcription in chondrocytes, however, remain largely unknown. Sox9 enhancers have been identified for developing gonad, neural crest, and spinal cord, but none have been discovered for chondrocytes. The overarching goal of the research project is to identify the mechanisms underlying Sox9 expression in chondrocytes. We have identified a highly conserved enhancer located far upstream of the Sox9 promoter. Preliminary results in transgenic mice indicate that this enhancer is active in proliferative chondrocytes in cartilage primordia and growth plates. I propose that this enhancer binds proteins that have a key role in ensuring Sox9 expression in these cells and are thereby required to maintain chondrocyte differentiation and ensure skeletal growth and timely ossification. The main goal of this project is to test this hypothesis by pursuing the following three specific aims: (1) define the domain of activity of the enhancer in vivo, (2) test whether th enhancer is required for Sox9 expression in vivo, and (3) identify the transcription factors that bind to and activate the Sox9 enhancer, and to determine their importance in chondrogenesis. Understanding the molecular basis of Sox9 expression in chondrogenesis will identify potential pharmacological therapeutic targets, which may have clinical implications for the treatment of skeletal dysplasias and degenerative cartilage diseases such as osteoarthritis.